ng quantities of the indicated peptides. Western blot analysis of MC491 using wild-type Dt40 cells and Dot1L-/- Dt40 cells. 28 H3K79me2 and H3S10ph were used as controls. MC491 and H3S10ph staining of HeLa cells with or without Hesperidin treatment. 442 Cell Cycle Volume 13 Issue 3 2014 Landes Bioscience. Do not distribute. line) cells all displayed a similar enrichment of H3T80ph in cells with a 4N DNA content. However, not all cells with a 4N DNA content were positive for H3T80ph, indicating that H3T80ph is unlikely to be enriched through all stages of G2 /M. Consequently, the precise spatialtemporal localization of H3T80ph during mitosis was examined via 
indirect immunofluorescence analysis in HeLa cells. H3T80ph was not detectable during interphase and became visible on chromatin in prophase. H3T80ph was maintained on the chromatin from prophase through metaphase, apparently being distributed throughout all regions of the chromosomes, but was no longer detectable in anaphase. In addition to the chromosomal staining of H3T80ph, H3T80ph is also apparent in the centrosomes during mitosis. Taken together, these data show that H3T80ph is present on chromatin from prophase to metaphase of mitosis. Histone H3 and the region around H3T80 are highly conserved, leading us to examine whether H3T80ph was also conserved in other eukaryotes. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19821986 We were unable to detect any H3T80ph in either budding yeast or fission yeast by immunofluorescence. However, H3T80ph was clearly present on the metaphase chromosomes of Drosophila S2 cells and from prophase to early anaphase in mouse MM3MG cells. In the MM3MG cells, the H3T80ph levels were reduced by early anaphase and undetectable by late anaphase. Notably, H3T80ph foci were observed in MM3MG cells in the very early stages of mitosis that had already acquired H3S10ph. H3S10ph foci are known to initiate at the centromere before spreading throughout chromatin.5 To determine whether or not H3T80ph foci were also present at the centromeres, cells were examined for the coincidence of H3T80ph and H3S10ph foci. H3T80ph foci did not co-localize with the most intense H3S10ph staining at the centromeres, indicating that H3T80ph foci are not reflecting centromeric localization. The identity of the chromosomal localization of the H3T80ph foci in late G2 /early mitosis is not known, but this data indicates that H3T80ph does not initiate at pericentric heterochromatin, as is the case for H3S10ph and H3S28ph. This www.landesbioscience.com Cell Cycle 443 2014 Landes Bioscience. Do not distribute. 444 Cell Cycle Volume 13 Issue 3 2014 Landes Bioscience. Do not distribute. separation of localization underscores a potential separation of function for the H3S10ph and H3T80ph mitotic markers. Mutations in H3T80 result in mitotic phenotypes Because H3T80ph localizes to chromatin in mitosis, we asked whether loss of the phospho-regulation of histone H3 at this site might interfere with mitotic events. To do this, we transiently transfected, exogenous V5-His tagged wild-type and mutant histone H3T80 constructs into HeLa cells. The H3T80 mutations were designed to be either non-phosphorylatable H3S10ph staining merged with DApI. www.landesbioscience.com or to mimic KU55933 constitutive phosphorylation. After verifying that the WT and mutant tagged proteins were associated with chromatin, asynchronous cells were examined and categorized based on 3 parameters: the phase of mitosis; whether unaligned chromatin was present at metaphase; and wh